Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/314—Phosphoramidates
  • C12N2310/3145—Phosphoramidates with the nitrogen in 3' or 5'-position
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/32—Chemical structure of the sugar
  • C12N2310/323—Chemical structure of the sugar modified ring structure
  • C12N2310/3233—Morpholino-type ring
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/33—Alteration of splicing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/35—Special therapeutic applications based on a specific dosage / administration regimen

Definitions

• 4140_032PC01_Seqlisting_ST25; Size: 12,950 bytes and Date of Creation: June 17, 2020) is herein incorporated by reference in its entirety.
• the disclosure relates to methods for treating muscular dystrophy in a human subject.
• DMD Duchenne Muscular Dystrophy
• DMD is a serious, progressively debilitating, and ultimately fatal inherited X-linked neuromuscular disease.
• DMD is caused by mutations in the dystrophin gene characterized by the absence, or near absence, of functional dystrophin protein that disrupt the mRNA reading frame, resulting in a lack of dystrophin, a critically important part of the protein complex that connects the cytoskeletal actin of a muscle fiber to the extracellular matrix.
• patients with DMD follow a predictable disease course.
• DMD remains an ultimately fatal disease, with patients usually dying of respiratory or cardiac failure in their mid to late 20s.
• a potential therapeutic approach to the treatment of DMD is suggested by Becker muscular dystrophy (BMD), a milder dystrophinopathy. Both dystrophinopathies are caused by mutations of the DMD gene.
• BMD Becker muscular dystrophy
• In DMD mutations that disrupt the pre-mRNA reading frame, referred to as "out-of-frame” mutations, prevent the production of functional dystrophin.
• in-frame mutations do not disrupt the reading frame and result in the production of internally shortened, functional dystrophin protein.
• An important approach for restoring these "out-of-frame" mutations is to utilize an antisense oligonucleotide to exclude or skip the molecular mutation of the DMD gene (dystrophin gene).
• the DMD or dystrophin gene is one of the largest genes in the human body and consists of 79 exons.
• Antisense oligonucleotides (AONs) have been specifically designed to target specific regions of the pre-mRNA, typically exons to induce the skipping of a mutation of the DMD gene thereby restoring these out-of-frame mutations in-frame to enable the production of internally shortened, yet functional dystrophin protein.
• Exondys 51 ® is a phosphorodiamidate morpholino oligomer (PMO) designed to skip exon 51 of the human dystrophin gene in patients with DMD who are amenable to exon 51 skipping to restore the read frame and produce a functional shorter form of the dystrophin protein.
• PMO phosphorodiamidate morpholino oligomer
• the recommended dose of EXONDYS 51 ® is 30 mg/kg administered once weekly as a 35 to 60 minute intravenous infusion.
• dystrophy such as DMD and BMD, in patients.
• the present disclosure provides methods of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g. eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg/kg.
• eteplirsen is administered at a dose of about 80 to about 300 mg/kg.
• golodirsen is administered at a dose of about 80 to about 300 mg/kg.
• casimersen is administered at a dose of about 80 to about 300 mg/kg.
• the present disclosure provides a method of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg/kg.
• eteplirsen is administered at a dose of about 100 mg/kg.
• golodirsen is administered at a dose of about 100 mg/kg.
• casimersen is administered at a dose of about 100 mg/kg.
• the present disclosure provides a method of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg/kg.
• eteplirsen is administered at a dose of about 100 mg/kg.
• golodirsen is administered at a dose of about 100 mg/kg.
• casimersen is administered at a dose of about 100 mg/kg.
• the present disclosure provides a method of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg/kg.
• eteplirsen is administered at a dose of about 200 mg/kg.
• golodirsen is administered at a dose of about 200 mg/kg.
• casimersen is administered at a dose of about 200 mg/kg.
• the present disclosure provides a method of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg/kg.
• eteplirsen is administered at a dose of about 200 mg/kg.
• golodirsen is administered at a dose of about 200 mg/kg.
• casimersen is administered at a dose of about 200 mg/kg.
• the present disclosure provides a method of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg/kg.
• eteplirsen is administered at a dose of about 300 mg/kg.
• golodirsen is administered at a dose of about 300 mg/kg. In one embodiment of the method, casimersen is administered at a dose of about 300 mg/kg.
• the present disclosure provides a method of treating DMD in a human subject having a mutation of the DMD gene that is amendable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, comprising administering to the human subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg/kg.
• eteplirsen is administered at a dose of about 300 mg/kg.
• golodirsen is administered at a dose of about 300 mg/kg.
• casimersen is administered at a dose of about 300 mg/kg.
• the disclosure also relates to methods of restoring an mRNA reading frame to induce dystrophin production in a human subject having a mutation of the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg/kg.
• eteplirsen is administered at a dose of about 80 to about 300 mg/kg. In one embodiment of the method, golodirsen is administered at a dose of about 80 to about 300 mg/kg. In one embodiment of the method, casimersen is administered at a dose of about 80 to about 300 mg/kg.
• the disclosure provides a method of restoring an mRNA
• the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg/kg.
• eteplirsen is administered at a dose of about 100 mg/kg.
• golodirsen is administered at a dose of about 100 mg/kg.
• casimersen is administered at a dose of about 100 mg/kg.
• the disclosure provides a method of restoring an mRNA reading frame to induce dystrophin production in a human subject having a mutation of the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg/kg.
• eteplirsen is administered at a dose of about 200 mg/kg. In one embodiment of the method, golodirsen is administered at a dose of about 200 mg/kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg/kg.
• the disclosure provides a method of restoring an mRNA
• the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg/kg.
• eteplirsen is administered at a dose of about 300 mg/kg.
• golodirsen is administered at a dose of about 300 mg/kg.
• casimersen is administered at a dose of about 300 mg/kg.
• the present disclosure also relates a method of excluding 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation of the dystrophin gene that is amenable to 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg/kg.
• eteplirsen is administered at a dose of about 80 to about 300 mg/kg.
• golodirsen is administered at a dose of about 80 to about 300 mg/kg.
• casimersen is administered at a dose of about 80 to about 300 mg/kg.
• the disclosure provides a method of excluding exon 44, exon
• the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg/kg.
• eteplirsen is administered at a dose of about 100 mg/kg. In one embodiment of the method, golodirsen is administered at a dose of about 100 mg/kg. In one embodiment of the method, casimersen is administered at a dose of about 100 mg/kg.
• the disclosure provides a method of excluding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation of the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg/kg. In one embodiment of the method, eteplirsen is administered at a dose of about 200 mg/kg. In one embodiment of the method, golodirsen is administered at a dose of about 200 mg/kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg/kg.
• the disclosure provides a method of excluding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 from dystrophin pre-mRNA during mRNA processing in a human subject having a mutation of the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg/kg. In one embodiment of the method, eteplirsen is administered at a dose of about 300 mg/kg. In one embodiment of the method, golodirsen is administered at a dose of about 300 mg/kg. In one embodiment of the method, casimersen is administered at a dose of about 200 mg/kg.
• the disclosure provides a method of binding exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of dystrophin pre-mRNA in a human subject having a mutation of the dystrophin gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 80 to about 300 mg/kg.
• eteplirsen is administered at a dose of about 80 to about 300 mg/kg.
• golodirsen is administered at a dose of about 80 to about 300 mg/kg.
• casimersen is administered at a dose of about 80 to about 300 mg/kg.
• the disclosure provides a method of binding exon 44, exon
• the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 100 mg/kg.
• eteplirsen is administered at a dose of about 100 mg/kg.
• golodirsen is administered at a dose of about 100 mg/kg.
• casimersen is administered at a dose of about 100 mg/kg.
• the disclosure provides a method of binding exon 44, exon
• the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 200 mg/kg.
• eteplirsen is administered at a dose of about 200 mg/kg.
• golodirsen is administered at a dose of about 200 mg/kg.
• casimersen is administered at a dose of about 200 mg/kg.
• the disclosure provides a method of binding exon 44, exon
• the method comprising administering to the subject a pharmaceutical composition comprising an antisense oligomer, or
• eteplirsen, golodirsen, or casimersen is administered at a dose of about 300 mg/kg.
• eteplirsen is administered at a dose of about 300 mg/kg.
• golodirsen is administered at a dose of about 300 mg/kg.
• casimersen is administered at a dose of about 300 mg/kg.
• the methods of the present disclosure comprise
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., etepbrsen
• an antisense oligomer e.g., golodirsen
• an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, or about 300 mg/kg.
• an antisense oligomer, or pharmaceutically acceptable salt thereof, (e.g., etepbrsen) is administered at a dose of about 100 mg/kg.
• an antisense oligomer (e.g., etepbrsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• the methods of the present disclosure comprise
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., etepbrsen), or pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., etepbrsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimeren), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• an antisense oligomer e.g., casimeren
• pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• the methods of the present disclosure comprise
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimeren), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• an antisense oligomer e.g., casimeren
• pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• the methods of the present disclosure comprise
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimeren), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• an antisense oligomer e.g., casimeren
• pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg/kg, wherein the pharmaceutical composition is administered once weekly.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• an antisense oligomer e.g., eteplirsen
• the methods of the present disclosure comprise
• a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, or up to about 200 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered for up to about 144 weeks.
• the composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered for the duration of the illness.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof e.g., eteplirsen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• an antisense oligomer e.g., eteplirsen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., golodirsen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., golodirsen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• an antisense oligomer e.g., golodirsen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., casimersen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., casimersen
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen) or pharmaceutically acceptable salt thereof, for up to about 48 weeks, wherein the antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• a method comprises administering to the human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 144 weeks, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 300 mg/kg.
• an antisense oligomer e.g., casimersen
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is formulated for systemic administration.
• the composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered intravenously.
• the composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered as an intravenous infusion.
• the pharmaceutical composition comprising an antisense oligomer e.g., eteplirsen, or pharmaceutically acceptable salt thereof
• the composition comprising an antisense oligomer e.g., eteplirsen
• the composition comprising an antisense oligomer e.g., eteplirsen or pharmaceutically acceptable salt thereof, is administered orally.
• the methods of the present disclosure comprise
• an antisense oligomer e.g., eteplirsen
• a male human subject wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer is administered at a dose of about 100 mg/kg.
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered at a dose of 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• a pharmaceutically acceptable salt thereof is administered at a dose of 300 mg/kg.
• the human subject is 7 to 13 years of age (inclusive).
• the methods of the present disclosure comprise
• an antisense oligomer e.g., golodirsen
• a male human subject wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• an antisense oligomer e.g., golodirsen
• an antisense oligomer e.g., golodirsen
• an antisense oligomer e.g., golodirsen
• pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., golodirsen
• an antisense oligomer e.g., golodirsen
• the pharmaceutically acceptable salt thereof is administered at a dose of 300 mg/kg.
• the human subject is 7 to 13 years of age (inclusive).
• the methods of the present disclosure comprise
• an antisense oligomer e.g., casimersen
• a male human subject wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• an antisense oligomer e.g., casimersen
• an antisense oligomer e.g., casimersen
• an antisense oligomer e.g., casimersen
• pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., casimersen
• an antisense oligomer e.g., casimersen
• the pharmaceutically acceptable salt thereof is administered at a dose of 300 mg/kg.
• the human subject is 7 to 13 years of age (inclusive).
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, further comprises a pharmaceutically acceptable carrier.
• the pharmaceutically acceptable carrier is a saline solution that includes a phosphate buffer, e.g., a phosphate- buffered saline.
• the methods of the present disclosure increase the number of dystrophin-positive fibers in the human subject.
• the human subjects treated with the methods of the disclosure achieve a higher North American Ambulatory Assessment (NSAA) total score.
• NSAA North American Ambulatory Assessment
• the higher NSAA score is achieved at about week 24, about week 48, about week 60, about week 80, about week 100, about week 120, about week 140, about week 150, about week 160, about week 180, or about week 200, relative to baseline.
• the higher NSAA score is achieved at about week 144, relative to baseline.
• the higher NSAA score is achieved at about week 164, relative to baseline.
• the methods of the disclosure reduce loss of ambulation, relative to baseline in the human subject, as measured by the 6 Minute Walk Test (6MWT).
• ambulation is maintained, relative to baseline.
• ambulation is improved, relative to baseline.
• the methods of the disclosure reduces loss of
• the loss of pulmonary function can be measured as % annual decline rate in Forced Vital Capacity (FVC).
• FVC Forced Vital Capacity
• the methods of the disclosure include administering
• Another therapeutic agent such as a steroid, to the subject.
• the methods of the disclosure comprise administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, as an intravenous infusion at a dose of about 100 mg/kg once weekly for up to 24 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise
• administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, as an intravenous infusion at a dose of about 100 mg/kg once weekly for up to 48 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, as an intravenous infusion at a dose of about 100 mg/kg once weekly for up to 144 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, administered as an intravenous infusion at a dose of about 200 mg/kg once weekly for up to 24 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise
• administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligonucleotide, administered as an intravenous infusion at a dose of about 200 mg/kg once weekly for up to 48 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise
• administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligonucleotide administered as an intravenous infusion at a dose of about 300 mg/kg once weekly for up to 48 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, administered as an intravenous infusion at a dose of about 200 mg/kg once weekly for up to 144 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the methods of the disclosure comprise administering to a human subject with a DMD having a mutation of the DMD gene that is amenable to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, administered as an intravenous infusion at a dose of about 300 mg/kg once weekly for up to 144 weeks.
• the human subject is male of 7 to 13 years of age (inclusive).
• the antisense oligomer is eteplirsen, golodirsen, or casimersen.
• the patent or application file contains at least one drawing executed in color.
• Figure 1 depicts increase in % exon 51 skipping in hDMD D52 mdx mice following administration of high-dose eteplirsen.
• Figure 2 depicts dystrophin production in hDMD D52 mdx mice following
• Figure 3 depicts results of grip strength test in hDMD D52 mdx mice
• Figure 4 depicts exon skipping in non-human primates (NHP) in quadriceps, heart, and diaphragm following administration of high-dose eteplirsen.
• Figure 5 compares exon skipping in NHPs in quadriceps following intravenous or subcutaneous administration of high-dose eteplirsen.
• Figure 6 provides images of stained myotube cultures treated with increasing concentrations of golodirsen (with myosin heacy chain in red and dystrophin in green).
• Figure 7 provides high content analysis results of the dystrophin staining intensity measures at different treatment concentration.
• Embodiments of the present invention relate to improved methods for treating muscular dystrophy, such as DMD and BMD, by administering antisense compounds that are specifically designed to induce exon skipping in the human dystrophin gene.
• Dystrophin plays a vital role in muscle function, and various muscle-related diseases are characterized by mutated forms of this gene.
• the improved methods described herein may be used for inducing exon skipping in mutated forms of the human dystrophin gene, such as the mutated dystrophin genes found in DMD and BMD.
• the antisense compounds of the present invention hybridize to selected regions of a pre-processed RNA of a mutated human dystrophin gene, induce exon skipping and differential splicing in that otherwise aberrantly spliced dystrophin mRNA, and thereby allow muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein.
• the resulting dystrophin protein is not necessarily the "wild-type" form of dystrophin, but is rather an internally truncated, yet functional or semi-functional, form of dystrophin.
• these and related embodiments are useful in the prophylaxis and treatment of muscular dystrophy, especially those forms of muscular dystrophy, such as DMD and BMD, that are characterized by the expression of defective dystrophin proteins due to aberrant mRNA splicing.
• the methods described herein further provide improved treatment options for patients with muscular dystrophy and offer significant and practical advantages over alternate methods of treating relevant forms of muscular dystrophy.
• the improved methods relate to the administration of an antisense compound for inducing exon skipping in the human dystrophin gene at a higher dose and/or for a longer duration than prior approaches.
• the invention relates to improved methods for treating muscular dystrophy such as DMD and BMD, by inducing exon skipping in a patient.
• exon skipping is induced by administering an effective amount of a composition which includes a charge-neutral, phosphorodiamidate morpholino oligomer (PMO), such as eteplirsen, which selectively binds to a target sequence in an exon of dystrophin pre- mRNA.
• PMO charge-neutral, phosphorodiamidate morpholino oligomer
• the invention relates to methods of treating DMD or BMD in which an effective amount of a composition e.g., about 80 mg/kg to about 300 mg/kg, which includes an antisense oligomer, or pharmaceutically acceptable salt thereof, as described herein, such as eteplirsen, over a period of time sufficient to treat the disease.
• a composition e.g., about 80 mg/kg to about 300 mg/kg, which includes an antisense oligomer, or pharmaceutically acceptable salt thereof, as described herein, such as eteplirsen
• the antisense oligomer, or pharmaceutically acceptable salt thereof is complementary to one or more exons or a portion thereof in the transcript.
• the one or more exons or a portion thereof are selected from group consisting of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53, and any combination thereof.
• the antisense oligomer, or pharmaceutically acceptable salt thereof is complementary to one or more exons or a portion thereof in the transcript.
• the one or more exons or a portion thereof are selected from group consisting of exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53, and any combination thereof.
• pharmaceutically acceptable salt thereof induces skipping of exon 51, exon 45, or exon 53 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 53 of the dystrophin transcript. In some embodiments, the antisense oligomer, or pharmaceutically acceptable salt thereof, induces skipping of exon 45 of the dystrophin transcript. In some embodiments, the antisense oligomer, or pharmaceutically acceptable salt thereof, induces skipping of exon 44 of the dystrophin transcript. In some embodiments, the antisense oligomer, or pharmaceutically acceptable salt thereof, induces skipping of exon 50 of the dystrophin transcript. In some
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 51 of the dystrophin transcript. In some embodiments, the antisense oligomer, or pharmaceutically acceptable salt thereof, induces skipping of exon 52 of the dystrophin transcript.
• the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer, or pharmaceutically acceptable salt thereof, the antisense oligomer, or pharmaceutically acceptable salt thereof, is complementary to one or more exons or a portion thereof in the dystrophin transcript.
• the one or more exons or a portion thereof are selected from the group consisting of exon 44, exon 45, exon 50, exon 51, exon 52, exon 53.
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 51, exon 45, or exon 53 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 51 of the dystrophin transcript. In some embodiments, the antisense oligomer, or pharmaceutically acceptable salt thereof, induces skipping of exon 45 of the dystrophin transcript. In some embodiments, the antisense oligomer, or pharmaceutically acceptable salt thereof, induces skipping of exon 53 of the dystrophin transcript.
• Various mutations in the dystrophin gene are amenable to exon 51 skipping.
• Non limiting examples of mutations in the following exons are amenable to exon 51 skipping include, e.g.: 45-50, 47-50, 48-50, 49-50, 50, 52, 52-63 (Leiden Duchenne muscular dystrophy mutation database, Leiden University Medical Center, The Netherlands). Determining whether a patient has a mutation in the DMD gene that is amenable to exon skipping is well within the purview of one of skill in the art (see, e.g., Aartsma-Rus et al. (2009) Hum Mut 30:293-299).
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 51 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof comprises a base sequence that is complementary to an exon 51 target region of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from:
• T and U of each of SEQ ID NOS: 1-6 are each thymine or uracil.
• the T and U in the antisense oligomer are both thymine.
• the T and U in the antisense oligomer are both uracil.
• the annealing site is H51A(+66+95).
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 53 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof comprises a base sequence that is complementary to an exon 53 target region of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from:
• T and U of each of SEQ ID NOS: 7-16 are each thymine or uracil.
• the T and U in the antisense oligomer are both thymine.
• the T and U in the antisense oligomer are both uracil.
• the annealing site is H53A(+36+60). In certain embodiments, the annealing site is H53A(+36+56).
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 45 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof comprises a base sequence that is complementary to an exon 45 target region of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from:
• T and U of each of SEQ ID NOS: 18-34 are each thymine or uracil.
• the T and U in the antisense oligomer are both thymine.
• the T and U in the antisense oligomer are both uracil.
• the annealing site is H45A(-03+19).
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 44 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof comprises a base sequence that is complementary to an exon 44 target region of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from:
• T of each of SEQ ID NOS: 35-41 is thymine or uracil.
• the T in the antisense oligomer is thymine. In certain embodiments, the T in the antisense oligomer is uracil.
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 50 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof comprises a base sequence that is complementary to an exon 50 target region of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from:
• T of each of SEQ ID NOS: 42-50 is thymine or uracil.
• the T in the antisense oligomer is thymine. In certain embodiments, the T in the antisense oligomer is uracil.
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces skipping of exon 52 of the dystrophin transcript.
• the antisense oligomer, or pharmaceutically acceptable salt thereof comprises a base sequence that is complementary to an exon 52 target region of the dystrophin transcript designated as an annealing site, wherein the base sequence and annealing site are selected from: wherein T of SEQ ID NO: 51 is thymine or uracil.
• the T in the antisense oligomer is thymine.
• the T in the antisense oligomer is uracil.
• the antisense oligomer is according to Formula (I):
• each Nu is a nucleobase which taken together form a targeting sequence; T is defined below; each Nu from 1 to (n+1) and 5' to 3' corresponds to the nucleobases in one of the following:
• each U and T in the antisense oligomer is independently thymine or uracil. In some embodiments, each T and U in the antisense oligomer is thymine.
• the annealing site is H51A(+66+95),
• T moiety attached to the 5' end of the antisense oligomer of Formula (I) is selected from:
• the methods comprise administering eteplirsen [sequence 5'-
• CTCCAACATCAAGGAAGATGGCATTTCTAG-3' (SEQ ID NO: l), a PMO designed to skip exon 51 of the human dystrophin gene in patients with DMD who are amenable to exon 51 skipping to restore the reading frame and produce a functional shorter form of the dystrophin protein.
• Eteplirsen is registered under CAS Registry Number 1173755-55- 9.
• Chemical names include: RNA, [P-deoxy-P-(dimethylamino)](2',3'-dideoxy-2',3'- imino-2',3'-seco)(2'a 5')(C-m5U- C-C-A-A-C-A-m5U-C-A-A-G-G-A-A-G-A-m5U-G- G-C-A-m5U-m5U-C-m5U-A-G), 5 [P- [4- [ [2- [2-(2- hydroxy ethoxy )ethoxy] ethoxy] carbonyl] - 1 -piperaziny 1] -A, A- dimethylphosphonamidate] and P,2',3'-trideoxy-P-(dimethylamino)-5'-O- ⁇ P-[4-(10-hydroxy-2,5,8- trioxadecanoyl)piperazin-l-yl]-
• the methods comprise administering golodirsen also known by its code name "SRP-4053.”
• Golodirsen is a PMO having the base sequence 5'- GTTGCCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO: 7).
• Golodirsen is registered under CAS Registry Number 1422959-91-8.
• Chemical names include: all-P-ambo- [P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a®5')(G-T-T-G-C-C-T-C- C-G-G-T-T-C-T-G-A-A-G-G-T-G-T-T-C) 5'-[4-( ⁇ 2-[2-(2- hydroxy ethoxy )ethoxy] ethoxy ⁇ carbonyl )-N.N-d ⁇ methyl pi pera/i ne- 1 -phosphonamidate] .
• Golodirsen's structure is depicted as following::
• the antisense oligomer is casimersen also known by its code name "SPR-4045" is a PMO having the base sequence 5'-
• CAATGCCATCCTGGAGTTCCTG - 3' (SEQ ID NO: 17). Casimersen is registered under CAS Registry Number 1422959-91-8. Chemical names include: all-P-ambo- [P,2',3'-trideoxy-P-(dimethylamino)-2',3'-imino-2',3'-seco](2'a®5')(C-A-A-T-G-C-C-A- T-C-C-T-G-G-A-G-T-T-C-T-G) 5'-[4-( ⁇ 2-[2-(2- hydroxy ethoxy )ethoxy] ethoxy ⁇ carbonyl)-N,N-dimethylpiperazine- 1 -phosphonamidate] .
• Embodiments of the disclosure relate to methods for treating muscular dystrophy, such as DMD and BMD, by administering an antisense oligomer, or pharmaceutically acceptable salt thereof, either per se or as a pharmaceutical composition.
• Dystrophin plays a vital role in muscle function, and various muscle-related diseases are
• the methods described herein may be used for inducing exon skipping in mutated forms of the human dystrophin gene, such as the mutated dystrophin genes found in DMD and BMD.
• these mutated human dystrophin genes either express defective dystrophin protein or express no measurable dystrophin at all, a condition that leads to various forms of muscular dystrophy.
• an antisense oligomer hybridizes to exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 of a pre-processed RNA of a mutated human dystrophin gene, induces exon skipping and differential splicing in that otherwise aberrantly spliced dystrophin mRNA, and thereby allows muscle cells to produce an mRNA transcript that encodes a functional dystrophin protein.
• the resulting dystrophin protein is not necessarily the "wild-type" form of dystrophin, but is rather a truncated, yet functional or semi-functional, form of dystrophin.
• these and related embodiments are useful in the prophylaxis and treatment of muscular dystrophy, especially those forms of muscular dystrophy, such as DMD and BMD, that are characterized by the expression of defective dystrophin proteins due to aberrant mRNA splicing.
• the methods described herein further provide treatment options for patients with muscular dystrophy and offer significant and practical advantages over alternate methods of treating relevant forms of muscular dystrophy.
• the methods relate to the administration of an antisense oligomer, or pharmaceutically acceptable salt thereof, for inducing exon skipping in the human dystrophin gene at a higher dose and/or for a longer duration than prior approaches.
• the disclosure relates to methods for treating muscular dystrophy such as
• exon skipping is induced by administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• an antisense oligomer e.g., eteplirsen
• the disclosure relates to methods of treating DMD or BMD, comprising administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, or about 300 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg/kg.
• exon skipping is induced by administering a
• composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• the disclosure relates to methods of treating DMD or BMD, comprising administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, or about 300 mg/kg.
• an antisense oligomer e.g., golodirsen
• an antisense oligomer e.g., golodirsen
• a pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., golodirsen
• exon skipping is induced by administering a
• composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• an antisense oligomer e.g., casimersen
• pharmaceutically acceptable salt thereof wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 80 to about 300 mg/kg.
• the disclosure relates to methods of treating DMD or BMD, comprising administering to a human subject a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or pharmaceutically acceptable salt thereof, is administered at a dose of about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, or about 300 mg/kg.
• an antisense oligomer e.g., casimersen
• pharmaceutically acceptable salt thereof is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., casimersen
• pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg/kg.
• the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, once weekly.
• a pharmaceutical composition comprising an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered twice weekly.
• the composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered once monthly.
• the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, once weekly.
• the composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof is administered twice weekly.
• the composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof is administered once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof is administered once monthly.
• the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, once weekly.
• the composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof is administered twice weekly.
• the composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof is administered once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof is administered once monthly.
• the methods comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, up to about 200 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered for up to about 144 weeks.
• the composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered for the duration of the illness.
• the methods comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, up to about 200 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof is administered for up to about 144 weeks.
• the composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, is administered for the duration of the illness.
• the methods comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, for up to about 24 weeks, up to about 48 weeks, up to about 60 weeks, up to about 80 weeks, up to about 100 weeks, up to about 120 weeks, up to about 140 weeks, up to about 150 weeks, up to about 160 weeks, up to about 180 weeks, up to about 200 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof is administered for up to about 144 weeks.
• the composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof is administered for at least 24 weeks, at least 36 weeks, at least 48 weeks, at least 120 weeks, at least 144 weeks, or at least 164 weeks.
• the pharmaceutical composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof is administered for the duration of the illness.
• “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
• antisense oligomer and "antisense compound” and "antisense
• oligonucleotide are used interchangeably and refer to a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid: oligomer heteroduplex within the target sequence.
• a nucleic acid typically an RNA
• Watson-Crick base pairing to form a nucleic acid: oligomer heteroduplex within the target sequence.
• morpholino oligomer or "PMO” (phosphoramidate- or
• phosphorodiamidate morpholino oligomer refer to an oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged or cationic joining the morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide.
• the synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, 5,506,337, 8,076,476, 8,299,206 and 7,943,762
• An "exon” refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after either portions of a pre-processed (or precursor) RNA have been removed by splicing.
• the mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA.
• mRNA messenger RNA
• rRNA rRNA
• tRNA tRNA
• the human dystrophin gene has about 79 exons.
• an "intron” refers to a nucleic acid region (within a gene) that is not translated into a protein.
• An intron is a non-coding section that is transcribed into a precursor mRNA (pre-mRNA), and subsequently removed by splicing during formation of the mature RNA.
• “Exon skipping” refers generally to the process by which an entire exon, or a portion thereof, is removed from a given pre-processed RNA, and is thereby excluded from being present in the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein, typically creating an altered, though still functional, form of the protein.
• Dystrophin is a rod-shaped cytoplasmic protein, and a vital part of the protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane.
• Dystrophin contains multiple functional domains. For instance, dystrophin contains an actin binding domain at about amino acids 14-240 and a central rod domain at about amino acids 253-3040. This large central domain is formed by 24 spectrin-like triple-helical elements of about 109 amino acids, which have homology to alpha-actinin and spectrin.
• the repeats are typically interrupted by four proline-rich non-repeat segments, also referred to as hinge regions.
• Repeats 15 and 16 are separated by an 18 amino acid stretch that appears to provide a major site for proteolytic cleavage of dystrophin.
• the sequence identity between most repeats ranges from 10- 25%.
• One repeat contains three alpha-helices: 1, 2 and 3.
• Alpha-helices 1 and 3 are each formed by 7 helix turns, probably interacting as a coiled-coil through a hydrophobic interface.
• Alpha-helix 2 has a more complex structure and is formed by segments of four and three helix turns, separated by a Glycine or Proline residue.
• Each repeat is encoded by two exons, typically interrupted by an intron between amino acids 47 and 48 in the first part of alpha-helix 2. The other intron is found at different positions in the repeat, usually scattered over helix-3.
• Dystrophin also contains a cysteine-rich domain at about amino acids 3080-3360), including a cysteine-rich segment (i.e., 15 Cysteines in 280 amino acids) showing homology to the C-terminal domain of the slime mold
• the carboxy-terminal domain is at about amino acids 3361-3685.
• the amino-terminus of dystrophin binds to F-actin and the carboxy-terminus binds to the dystrophin-associated protein complex (DAPC) at the sarcolemma.
• the DAPC includes the dystroglycans, sarcoglycans, integrins and caveolin, and mutations in any of these components cause autosomally inherited muscular dystrophies.
• the DAPC is destabilized when dystrophin is absent, which results in diminished levels of the member proteins, and in turn leads to progressive fibre damage and membrane leakage.
• muscle cells produce an altered and functionally defective form of dystrophin, or no dystrophin at all, mainly due to mutations in the gene sequence that lead to incorrect splicing.
• a "defective" dystrophin protein may be characterized by the forms of dystrophin that are produced in certain subjects with DMD or BMD, as known in the art, or by the absence of detectable dystrophin.
• “Amenable to exon 53 skipping” as used herein with regard to a subject or patient is intended to include subjects and patients having one or more mutations in the dystrophin gene which, absent the skipping of exon 53 of the dystrophin gene, causes the reading frame to be out-of-frame thereby disrupting translation of the pre-mRNA leading to an inability of the subject or patient to produce dystrophin.
• Non-limiting examples of mutations in the following exons of the dystrophin gene are amenable to exon 53 skipping include, e.g., deletion of: exons 3 to 52, 4 to 52, 5 to 52, 6 to 52, 9 to 52, 10 to 52, 11 to 52, 13 to 52, 14 to 52, 15 to 52, 16 to 52, 17 to 52, 19 to 52, 21 to 52, 23 to 52, 24 to 52,
• “Amenable to exon 45 skipping” as used herein with regard to a subject or patient is intended to include subjects and patients having one or more mutations in the dystrophin gene which, absent the skipping of exon 45 of the dystrophin pre-mRNA, causes the reading frame to be out-of-frame thereby disrupting translation of the pre- mRNA leading to an inability of the subject or patient to produce functional or semi- functional dystrophin.
• mutations in the dystrophin gene that are amenable to exon 45 skipping include, e.g., mutations in exons 7-44, 12-44, 18-44, 44, 46, 46-47, 46- 48, 46-49, 46-51, 46-53, 46-55, 46-57, 46-59, 46-60, 46-67, 46-69, 46-75, and 46-78 (Leiden Duchenne muscular dystrophy mutation database, Leiden University Medical Center, The Netherlands). Determining whether a patient has a mutation in the dystrophin gene that is amenable to exon skipping is well within the purview of one of skill in the art (see, e.g., Aartsma-Rus et al. (2009) Hum Mutat. 30:293-299; Gurvich et al, Hum Mutat. 2009; 30(4) 633-640; and Fletcher et al. (2010) Molecular Therapy 18(6) 1218-1223).
• a "functional" dystrophin protein refers generally to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy, typically as compared to the altered or "defective" form of dystrophin protein that is present in certain subjects with DMD or BMD.
• a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art.
• dystrophin-related activity in muscle cultures in vitro can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al, Journal of Cell Science. 112:209-216, 1999).
• Animal models are also valuable resources for studying the pathogenesis of disease, and provide a means to test dystrophin-related activity.
• Two of the most widely used animal models for DMD research are the mdx mouse and the golden retriever muscular dystrophy (GRMD) dog, both of which are dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003).
• GRMD golden retriever muscular dystrophy
• These and other animal models can be used to measure the functional activity of various dystrophin proteins. Included are truncated forms of dystrophin, such as those forms that are produced by certain of the exon-skipping antis
• phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• pharmaceutically-acceptable carrier means a non toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.
• materials which can serve as pharmaceutically acceptable carriers are: sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar;
• buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; coating agents; sweetening agents; flavoring agents; perfuming agents; preservatives; and antioxidants; according to the judgment of the formulator.
• buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; coating agents; sweetening agents; flavoring agents; perfuming agents; preservatives; and antioxidants; according to the judgment of the formulator.
• the term "restoration" of dystrophin synthesis or production refers generally to the production of a dystrophin protein including truncated forms of dystrophin in a patient with muscular dystrophy following treatment with an antisense oligonucleotide as described herein.
• treatment results in an increase in novel dystrophin production in a patient.
• treatment increases the number of dystrophin-positive fibers of normal in the subject.
• the percent of dystrophin-positive fibers in a patient following treatment can be determined by a muscle biopsy using known techniques. For example, a muscle biopsy may be taken from a suitable muscle, such as the biceps brachii muscle in a patient.
• Analysis of the percentage of positive dystrophin fibers may be performed pre treatment and/or post-treatment or at time points throughout the course of treatment.
• a post-treatment biopsy is taken from the contralateral muscle from the pre-treatment biopsy.
• Pre- and post-treatment dystrophin expression studies may be performed using any suitable assay for dystrophin. In one embodiment,
• immunohistochemical detection is performed on tissue sections from the muscle biopsy using an antibody that is a marker for dystrophin, such as a monoclonal or a polyclonal antibody.
• a marker for dystrophin such as a monoclonal or a polyclonal antibody.
• the MANDYS106 antibody can be used which is a highly sensitive marker for dystrophin. Any suitable secondary antibody may be used.
• the percent dystrophin-positive fibers are calculated by dividing the number of positive fibers by the total fibers counted. Normal muscle samples have 100% dystrophin-positive fibers. Therefore, the percent dystrophin positive fibers can be expressed as a percentage of normal. To control for the presence of trace levels of dystrophin in the pretreatment muscle as well as revertant fibers a baseline can be set using sections of pre-treatment muscles from each patient when counting dystrophin-positive fibers in post-treatment muscles. This may be used as a threshold for counting dystrophin-positive fibers in sections of post-treatment muscle in that patient.
• antibody-stained tissue sections can also be used for dystrophin quantification using Bioquant image analysis software (Bioquant Image Analysis Corporation, Milwaukee, TN). The total dystrophin fluorescence signal intensity can be reported as a percentage of normal.
• Western blot analysis with monoclonal or polyclonal anti-dystrophin antibodies can be used to determine the percentage of dystrophin positive fibers.
• the anti-dystrophin antibody NCL-Dysl from Novacastra may be used.
• the percentage of dystrophin-positive fibers can also be analyzed by determining the expression of the components of the sarcoglycan complex (b,g) and/or neuronal NOS.
• Treatment of an individual (e.g. a mammal, such as a human subject) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell.
• Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
• Treatment includes any desirable effect on the symptoms or pathology of a disease or condition associated with the dystrophin protein, as in certain forms of muscular dystrophy, and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated.
• prophylactic treatments which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.
• Treatment or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.
• alkyl refers to a saturated straight or branched hydrocarbon.
• the alkyl group is a primary, secondary, or tertiary hydrocarbon.
• the alkyl group includes one to ten carbon atoms, i.e., Ci to Cio alkyl.
• the alkyl group includes one to six carbon atoms, i.e., Ci to G, alkyl.
• the term includes both substituted and unsubstituted alkyl groups, including halogenated alkyl groups.
• the alkyl group is a fluorinated alkyl group.
• Non-limiting examples of moieties with which the alkyl group can be substituted are selected from the group consisting of halogen (fluoro, chloro, bromo, or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al, Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference.
• halogen fluoro, chloro, bromo, or iodo
• hydroxyl amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or
• the alkyl group is selected from the group consisting of methyl, CF3, CCh, CFCb, CF2CI, ethyl, CH2CF3, CF2CF3, propyl, isopropyl, butyl, isobutyl, sec-butyl, t- butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.
• aryloxy refers to aromatic ring groups having six to fourteen ring atoms, such as phenyl, 1 -naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.
• An “aryl” ring may contain one or more substituents.
• the term “aryl” may be used interchangeably with the term “aryl ring.”
• “Aryl” also includes fused polycyclic aromatic ring systems in which an aromatic ring is fused to one or more rings. Non-limiting examples of useful aryl ring groups include phenyl, hydroxyphenyl, halophenyl, alkoxyphenyl,
• aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as in an indanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.
• acyl refers to a C(0)R group (in which R signifies H, alkyl or aryl as defined herein).
• R signifies H, alkyl or aryl as defined herein.
• acyl groups include formyl, acetyl, benzoyl, phenylacetyl and similar groups.
• oligomers i.e., each comprising a nucleobase sequence
• oligomers i.e., each comprising a nucleobase sequence
• target gene i.e., each comprising a target gene
• the nucleobase sequence "T-G-A (5'- 3') is complementary to the nucleobase sequence "A-C-T (3'- 5').”
• Complementarity may be "partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules.
• complementarity between a given nucleobase sequence and the other nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Or, there may be "complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example.
• the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
• treatment with the methods of the disclosure increases novel dystrophin production and slows or reduces the loss of ambulation that would be expected without treatment.
• treatment may stabilize, maintain, improve or increase walking ability (e.g., stabilization of ambulation) in the subject.
• treatment maintains or increases a stable walking distance in a patient, as measured by, for example, the 6 Minute Walk Test (6MWT), described by McDonald, et al. (Muscle Nerve, 2010; 42:966-74, herein incorporated by reference).
• a change in the 6 Minute Walk Distance (6MWD) may be expressed as an absolute value, a percentage change or a change in the %-predicted value.
• the performance of a DMD patient in the 6MWT relative to the typical performance of a healthy peer can be determined by calculating a %-predicted value.
• the %-predicted 6MWD may be calculated using the following equation for males: 196.72 + (39.81 x age) - (1.36 x age 2 ) + (132.28 x height in meters).
• the %-predicted 6MWD may be calculated using the following equation: 188.61 + (51.50 x age) - (1.86 x age 2 ) + (86.10 x height in meters) (Henricson et al. PLoS Curr., 2012, version 2, herein incorporated by reference).
• treatment with an antisense oligonucleotide increases the stable walking distance in the patient from baseline.
• Loss of muscle function in patients with DMD may occur against the background of normal childhood growth and development. Indeed, younger children with DMD may show an increase in distance walked during 6MWT over the course of about 1 year despite progressive muscular impairment.
• the 6MWD from patients with DMD is compared to typically developing control subjects and to existing normative data from age and sex matched subjects.
• normal growth and development can be accounted for using an age and height based equation fitted to normative data. Such an equation can be used to convert 6MWD to a percent- predicted (%-predicted) value in subjects with DMD.
• analysis of %-predicted 6MWD data represents a method to account for normal growth and development, and may show that gains in function at early ages (e.g., less than or equal to age 7) represent stable rather than improving abilities in patients with DMD (Henricson et al. PLoS Curr., 2012, version 2, herein incorporated by reference).
• treatment with an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, of the disclosure slows or reduces the progressive respiratory muscle dysfunction and/or failure in patients with DMD that would be expected without treatment.
• treatment with an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, of the disclosure may reduce or eliminate the need for ventilation assistance that would be expected without treatment.
• measurements of respiratory function for tracking the course of the disease, as well as the evaluation of potential therapeutic interventions include maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and forced vital capacity (FVC).
• MIP and MEP measure the level of pressure a person can generate during inhalation and exhalation, respectively, and are sensitive measures of respiratory muscle strength. MIP is a measure of diaphragm muscle weakness.
• MEP may decline before changes in other pulmonary artery
• MIP may be an early indicator of respiratory dysfunction.
• FVC may be used to measure the total volume of air expelled during forced exhalation after maximum inspiration. In patients with DMD, FVC increases concomitantly with physical growth until the early teens. However, as growth slows or is stunted by disease progression, and muscle weakness progresses, the vital capacity enters a descending phase and declines at an average rate of about 8 to 8.5 percent per year after 10 to 12 years of age.
• MIP percent predicted MIP adjusted for weight
• MEP percent predicted MEP adjusted for age
• FVC percent predicted FVC adjusted for age and height
• systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
• the disclosure relates to improved methods for treating muscular dystrophy such as DMD and BMD, by inducing exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping in a human subject.
• exon skipping is induced by
• an antisense oligomer, or pharmaceutically acceptable salt thereof is administered at a dose of about 80 to about 300 mg/kg.
• methods of the disclosure comprise administering a
• composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• an antisense oligomer e.g., eteplirsen
• eteplirsen an antisense oligomer
• pharmaceutically acceptable salt thereof is administered at doses of about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 105 mg/kg, about 110 mg/kg, about 120 mg/kg, about 125 mg/kg, about 130 mg/kg, about 135 mg/kg, about 140 mg/kg, about 145 mg/kg, about 150 mg/kg, about 155 mg/kg, about 160 mg/kg, about 165 mg/kg, about 170 mg/kg, about 175 mg/kg, about 180 mg/kg, about 185 mg/kg, about 190 mg/kg, about 195 mg/kg, about 200 mg/kg, about 205 mg/kg, about 210 mg/kg, about 215 mg/kg, about 220 mg/kg, about 225 mg/kg, about 230 mg/kg, about 235 mg/kg, about 240 mg/kg, about 245 mg/kg, about 250 mg/kg, about 255 mg/kg, about 260 mg/kg, about 265 mg/
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• the administration is via intravenous (i.v.) infusion.
• methods of the disclosure comprise administering a
• composition comprising an antisense oligomer (e.g., golodirsen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• pharmaceutically acceptable salt thereof is administered in doses from about 80 mg to about 300 mg per kilogram of body weight per day or about 100 mg to about 200 mg per kilogram of body weight per day. In some cases, doses of greater than 300 mg/kg may be necessary. In some embodiments, doses for administration are from about 80 mg to about 300 mg/kg.
• an antisense oligomer e.g., golodirsen
• an antisense oligomer is administered at doses of about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 105 mg/kg, about 110 mg/kg, about 120 mg/kg, about 125 mg/kg, about 130 mg/kg, about 135 mg/kg, about 140 mg/kg, about 145 mg/kg, about 150 mg/kg, about 155 mg/kg, about 160 mg/kg, about 165 mg/kg, about 170 mg/kg, about 175 mg/kg, about 180 mg/kg, about 185 mg/kg, about 190 mg/kg, about 195 mg/kg, about 200 mg/kg, about 205 mg/kg, about 210 mg/kg, about 215 mg/kg, about 220 mg/kg, about 225 mg/kg, about 230 mg/kg, about 235 mg/kg, about 240 mg/kg, about 245 mg//
• an antisense oligomer e.g., golodirsen
• a dose of about 100 mg/kg is administered at a dose of about 100 mg/kg.
• an antisense oligomer e.g., golodirsen
• a dose of about 200 mg/kg is administered at a dose of about 200 mg/kg.
• an antisense oligomer e.g., golodirsen
• pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg/kg.
• the administration is via intravenous (i.v.) infusion.
• methods of the disclosure comprise administering a
• composition comprising an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, wherein the antisense oligomer, or
• pharmaceutically acceptable salt thereof is administered in doses from about 80 mg to about 300 mg per kilogram of body weight per day or about 100 mg to about 200 mg per kilogram of body weight per day. In some cases, doses of greater than 300 mg/kg may be necessary. In some embodiments, doses for administration are from about 80 mg to about 300 mg/kg.
• an antisense oligomer (e.g., casimersen), or pharmaceutically acceptable salt thereof, is administered at doses of about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 105 mg/kg, about 110 mg/kg, about 120 mg/kg, about 125 mg/kg, about 130 mg/kg, about 135 mg/kg, about 140 mg/kg, about 145 mg/kg, about 150 mg/kg, about 155 mg/kg, about 160 mg/kg, about 165 mg/kg, about 170 mg/kg, about 175 mg/kg, about 180 mg/kg, about 185 mg/kg, about 190 mg/kg, about 195 mg/kg, about 200 mg/kg, about 205 mg/kg, about 210 mg/kg, about 215 mg/kg, about 220 mg/kg, about 225 mg/kg, about 230 mg/kg, about 235 mg/kg, about 240 mg/kg, about 245 mg/kg,
• an antisense oligomer e.g., casimersen
• an antisense oligomer e.g., casimersen
• an antisense oligomer e.g., casimersen
• the administration is via intravenous (i.v.) infusion.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be administered once every one, two, three, four or five years.
• dosing is one administration per day. In certain embodiments, dosing is one or more administration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2,
• dosing is one administration once every week. In some embodiments, dosing is one administration once every two weeks. In various embodiments, dosing is one or more administrations every month. In yet other embodiments, the antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.
• the antisense oligomer e.g., eteplirsen
• the antisense oligomer is administered once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, or once every 12 weeks.
• the pharmaceutical composition comprising an antisense oligomer e.g., eteplirsen, or pharmaceutically acceptable salt thereof, is administered once monthly.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be administered once every one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months.
• the pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered at regular intervals, e.g., daily; once every two days; once every three days; once every 3 to 7 days; once every 3 to 10 days; once every 7 to 10 days; once every week; once every two weeks; once monthly.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be administered once weekly by intravenous infusion.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be administered once monthly by intravenous infusion.
• An antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be administered intermittently over a longer period of time, e.g., for several weeks, months or years.
• the treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.
• an antisense oligomer e.g., eteplirsen
• eteplirsen e.g., eteplirsen
• composition is administered weekly at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered weekly at a dose of about 125 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered weekly at a dose of about 175 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer is administered weekly at a dose of about 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer is administered weekly at a dose of about 300 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof either per se or a pharmaceutical composition, is administered weekly at a dose of about 100 mg/kg via an intravenous infusion.
• an antisense oligomer e.g., eteplirsen
• a pharmaceutically acceptable salt thereof is administered weekly at a dose of about 200 mg/kg via an intravenous infusion.
• weekly is understood to have the art-accepted meaning of every week.
• an antisense oligomer e.g., eteplirsen
• eteplirsen e.g., eteplirsen
• composition is administered bi-weekly at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered bi-weekly at a dose of about 125 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered bi-weekly at a dose of about 175 mg/kg.
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered bi-weekly at a dose of about 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof either per se or a pharmaceutical composition
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, either per se or a pharmaceutical composition is administered bi-weekly at a dose of about 200 mg/kg via an intravenous infusion.
• biweekly is understood to have the art-accepted meaning of every two weeks.
• an antisense oligomer e.g., eteplirsen
• eteplirsen e.g., eteplirsen
• composition is administered every third week at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered every third week at a dose of about 125 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered every third week at a dose of about 150 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered every third week at a dose of about 200 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof either per se or a pharmaceutical composition
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, either per se or a pharmaceutical composition is administered every third week at a dose of about 200 mg/kg via an intravenous infusion.
• every third week is understood to have the art-accepted meaning of once every three weeks.
• an antisense oligomer e.g., eteplirsen
• eteplirsen e.g., eteplirsen
• composition is administered monthly at a dose of about 100 mg/kg.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered monthly at a dose of about 125 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is administered monthly at a dose of about 175 mg/kg.
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof either per se or a pharmaceutical composition
• monthly is understood to have the art-accepted meaning of every month.
• monthly administrations may be in one or more administrations or sub-doses as discussed herein.
• the time of intravenous infusion is from about 15 minutes to about 4 hours. In some embodiments, the time of infusion is from about 30 minutes to about 3 hours. In some embodiments, the time of infusion is from about 30 minutes to about 2 hours. In some embodiments, the time of infusion is from about 1 hour to about 2 hours. In some embodiments the time of infusion is from about 30 minutes to about 1 hour. In some embodiments, the time of infusion is about 60 minutes. In some embodiments, the time of infusion is 35 to 60 minutes.
• the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the composition is administered for up to about 24 weeks, up to about 25 weeks, up to about 26 weeks, up to about 27 weeks, up to about 28 weeks, up to about 29 weeks, up to about 30 weeks, up to about 31 weeks, up to about 32 weeks, up to about 33 weeks, up to about 34 weeks, up to about 35 weeks, up to about 36 weeks, up to about 37 weeks, up to about 38 weeks, up to about 39 weeks, up to about 40 weeks, up to about 41 weeks, up to about 42 weeks, up to about 43 weeks, up to about 44 weeks, up to about 45 weeks, up to about 46 weeks, up to about 47 weeks, up to about 48 weeks, up to about 49 weeks, up to about 50 weeks, up to about 51 weeks, up to about 52 weeks, up to about 53 weeks, up to about 54 weeks, up to about 55 weeks, up to about
• composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, is administered for up to about 48 weeks.
• pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered for up to about 144 weeks.
• pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof is administered for up to about 168 weeks.
• the methods of the disclosure comprise administering a pharmaceutical composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, wherein the composition is administered for at least about 24 weeks, at least about 25 weeks, at least about 26 weeks, at least about 27 weeks, at least about 28 weeks, at least about 29 weeks, at least about 30 weeks, at least about 31 weeks, at least about 32 weeks, at least about 33 weeks, at least about 34 weeks, at least about 35 weeks, at least about 36 weeks, at least about 37 weeks, at least about 38 weeks, at least about 39 weeks, at least about 40 weeks, at least about 41 weeks, at least about 42 weeks, at least about 43 weeks, at least about 44 weeks, at least about 45 weeks, at least about 46 weeks, at least about 47 weeks, at least about 48 weeks, at least about 49 weeks, at least about 50 weeks, at least about 51 weeks, at least about 52 weeks, at least about 53 weeks, at least about 54 weeks, at least about 55 weeks, at least about
• the composition comprising an antisense oligomer e.g., eteplirsen
• an antisense oligomer e.g., eteplirsen
• the pharmaceutical composition is administered for the duration of the illness.
• any of the methods described herein may involve treating a patient who has lost the ability to rise independently from supine.
• the patient loses the ability to rise independently from supine at least one year prior to treatment with an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof.
• an antisense oligomer e.g., eteplirsen
• the patient loses the ability to rise independently from supine within one year of beginning treatment with an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof. In some embodiments of any of the methods described herein, the patient loses the ability to rise independently from supine within two years of beginning treatment.
• an antisense oligomer e.g., eteplirsen
• any of the methods described herein comprise continuing treatment with an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, even if a patient loses the ability to rise from a supine position during treatment with the antisense oligomer, or pharmaceutically acceptable salt thereof.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof even if a patient loses the ability to rise from a supine position during treatment with the antisense oligomer, or pharmaceutically acceptable salt thereof.
• the patient has a rise time of greater than 10 seconds. In some embodiments of any of the methods described herein, the patient has a rise time of greater than 15 seconds. In some embodiments of any of the methods described herein, the patient has a rise time of greater than 20 seconds.
• the antisense oligomer, or pharmaceutically acceptable salt thereof induces exon 44, exon 45, exon 50, exon 51, exon 52, or exon 53 skipping, include percent dystrophin positive fibers (PDPF), six-minute walk test (6MWT), loss of ambulation (LOA), North Star Ambulatory Assessment (NSAA), pulmonary function tests (PFT), ability to rise (from a supine position) without external support, de novo dystrophin production, and other functional measures.
• PDPF percent dystrophin positive fibers
• 6MWT loss of ambulation
• NSAA North Star Ambulatory Assessment
• PFT pulmonary function tests
• ability to rise from a supine position without external support
• de novo dystrophin production and other functional measures.
• the methods of the disclosure delay progression of the disease in the human subject treated with the methods, as measured by the 6 Minute Walk Test (6MWT).
• 6MWT 6 Minute Walk Test
• the methods of the disclosure allow to maintain pulmonary function or reducing loss of pulmonary function in a human subject treated with the methods.
• pulmonary function is measured as Maximum Expiratory Pressure (MEP).
• MIP Maximum Inspiratory Pressure
• FVC Forced Vital Capacity
• the methods of the disclosure restore an mRNA reading frame to induce dystrophin protein production in a human subject with DMD.
• Protein production can be measured by reverse-transcription polymerase chain reaction (RT- PCR), western blot analysis, or immunohistochemistry (IHC).
• tissue from different muscle groups e.g., the quadriceps, diaphragm, biceps, skin, heart, etc.
• PCR for example, droplet digital PCR,“ddPCR”
• western blot analysis can be performed as described in Schnell et ah,“Challenges in Interpreting Dystrophin Content by Western Blot,” US Neurology, 2019;15(l):40-6, disclosure of which is incorporated herein in its entirety.
• the human subject to be treated by the methods of the disclosure is a male.
• the human subject e.g., male
• the human subject is between about 6 months and about 4 years of age, inclusive.
• the human subject is at least 6 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 36) months of age.
• the human subject is no greater than 4 years of age.
• the male human subject is 7 to 13 years of age (inclusive).
• the male human subject is younger than 7 years of age.
• the male human subject is over the age of 13.
• the methods of the disclosure also include administering a pharmaceutical
• composition comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, in combination with another therapeutic.
• additional therapeutic may be administered prior, concurrently or subsequently to the administration of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof.
• the additional therapeutic may be formulated in the same composition as the antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, or may be in a different composition.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be administered in combination with a steroid and/or an antibiotic.
• the steroid may be a glucocorticoid or prednisone.
• Other agents which can be administered include an antagonist of the ryanodine receptor, such as dantrolene, which has been shown to enhance antisense-mediated exon skipping in patient cells and a mouse model of DMD (G. Kendall et al. Sci Tranl Med 4 164ral60 (2012), incorporated herein by reference).
• the methods of the disclosure include co-administering an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, either per se or as a pharmaceutical composition, with a carbohydrate, either in the same composition or is a separate composition, as provided in Han et al. , Nat. Comms. 7,
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, may be co-administered with 5% of a hexose carbohydrate.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be co-administered with 5% glucose, 5% fructose, or 5% mannose.
• an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, may be co-administered with 2.5% glucose and 2.5% fructose.
• an antisense oligomer e.g., eteplirsen
• a carbohydrate selected from: arabinose present in an amount of 5% by volume, glucose present in an amount of 5% by volume, sorbitol present in an amount of 5% by volume, galactose present in an amount of 5% by volume, fructose present in an amount of 5% by volume, xylitol present in an amount of 5% by volume, mannose present in an amount of 5% by volume, a combination of glucose and fructose each present in an amount of 2.5% by volume, and a combination of glucose present in an amount of 5.7% by volume, fructose present in an amount of 2.86% by volume, and xylitol present in an amount of 1.4% by volume.
• an antisense oligomer e.g., eteplirsen
• eteplirsen e.g., eteplirsen
• the non-steroidal anti-inflammatory compound is an NF-kB inhibitor.
• the NF-kB inhibitor may be CAT- 1004 or a pharmaceutically acceptable salt thereof.
• the NF-kB inhibitor may be a conjugate of salicylate and DHA.
• the NF-kB inhibitor is CAT-1041 or a pharmaceutically acceptable salt thereof.
• the NF-kB inhibitor is a conjugate of salicylate and EPA.
• the NF-kB inhibitor is
• non-steroidal anti-inflammatory compound is a TGF-b inhibitor.
• the TGF-b inhibitor is HT-100.
• Methods according to the disclosure include administration of an antisense
• oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof as a
• composition comprising the antisense oligomer, or pharmaceutically acceptable salt thereof.
• Methods for the delivery of nucleic acid molecules are described, for example, in Akhtar et al, 1992, Trends Cell Bio., 2: 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al, PCT WO 94/02595. These and other protocols can be utilized for the delivery of virtually any nucleic acid molecule, including eteplirsen.
• the disclosure provides methods comprising
• compositions that comprise a
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
• the pharmaceutical composition comprises from about 0.1 to about 99% of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable excipient.
• the pharmaceutical composition comprises from about 1 to about 90% of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof.
• the pharmaceutical composition comprises from about 5 to about 70%, about 5 to about 60%, about 5 to about 50%, about 5 to about 40%, or about 5 to about 30% of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof.
• the pharmaceutical composition comprises from about 10 to about 80%, about 10 to about 70%, about 10 to about 60%, about 10 to about 50%, about 10 to about 40%, about 10 to about 30%, or about 10 to about 20% of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof.
• an antisense oligomer e.g., eteplirsen
• the concentration of an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof, in the pharmaceutical composition is about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, or about 200 mg/ml.
• concentration of an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof in the pharmaceutical composition
• compositions suitable for administration in the methods of the disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
• oral administration for example, drenches (aqueous or non-aqueous solutions
• Some examples of materials that can serve as pharmaceutically-acceptable carriers include, without limitation: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such
• antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P-gly coprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues; biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc.
• nanoparticles such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
• compositions comprising surface- modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, branched and unbranched or combinations thereof, or long-circulating liposomes or stealth liposomes).
• PEG-modified, branched and unbranched or combinations thereof, or long-circulating liposomes or stealth liposomes offer a method for increasing the accumulation of drugs in target tissues.
• This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).
• liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al, Science 1995, 267, 1275-1276; Oku et al, 1995, Biochim. Biophys. Acta, 1238, 86-90).
• the long- circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 1995, 42, 24864-24870; Choi et al, International PCT Publication No.
• WO 96/10391 Ansell et al., International PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No. WO 96/10392).
• Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
• the disclosure includes an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, compositions prepared for delivery as described in U.S. Pat. Nos. 6,692,911, 7,163,695 and 7,070,807.
• the disclosure provides an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, in a composition comprising copolymers of lysine and histidine (HK) (as described in U.S. Pat. Nos.
• the disclosure provides eteplirsen in compositions comprising gluconic-acid-modified polyhistidine or gluconylated-polyhistidine/transferrin-polylysine.
• PEG e.g., branched or unbranched PEG or a mixture of both
• an antisense oligomer e.g., eteplirsen
• a pharmaceutically acceptable salt refers to the relatively non-toxic, inorganic and organic acid and base addition salts of an antisense oligomer.
• Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and
• the pharmaceutically acceptable salts also include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids.
• such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
• Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like, are also included, as well as representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, e.g., Berge et al., supra).
• wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
• antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
• water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
• oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), le
• Formulations suitable for use in the methods of the disclosure include those
• the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
• the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety -nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
• Methods of preparing pharmaceutical compositions comprising an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof include the step of bringing into association an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, with the carrier and, optionally, one or more accessory ingredients.
• the formulations are prepared by uniformly and intimately bringing an antisense oligomer (e.g., etepbrsen), or pharmaceutically acceptable salt thereof, into association with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
• Formulations of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, as an active ingredient.
• An antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may also be administered as a bolus, electuary or paste.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically-acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example,
• compositions may also comprise buffering agents.
• Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared using binder (e.g., gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
• Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets, and other solid dosage forms of the pharmaceutical compositions of the disclosure may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example,
• hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
• Liquid dosage forms for oral administration of an antisense oligomer e.g.,
• eteplirsen include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifier
• the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
• Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• Formulations for rectal or vaginal administration may be presented as a
• suppository which may be prepared by mixing an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
• an antisense oligomer e.g., eteplirsen
• suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
• Formulations or dosage forms for the topical or transdermal administration of an antisense oligomer include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• An antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
• the ointments, pastes, creams and gels may contain, in addition to an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• an antisense oligomer e.g., eteplirsen
• excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• Powders and sprays can contain, in addition to an antisense oligomer (e.g.,
• Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• Transdermal patches have the added advantage of providing controlled delivery of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, to the body.
• an antisense oligomer e.g., eteplirsen
• Such dosage forms can be made by dissolving or dispersing the oligomer in the proper medium.
• Absorption enhancers can also be used to increase the flux of the agent across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the agent in a polymer matrix or gel, among other methods known in the art.
• compositions suitable for parenteral administration may comprise an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions
• aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as olive oil
• injectable organic esters such as ethyl oleate.
• the pharmaceutical composition comprises a phosphate-buffered saline.
• compositions may also contain adjuvants such as
• preservatives wetting agents, emulsifying agents and dispersing agents.
• Prevention of the action of microorganisms upon the subject oligomers may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
• prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. [0182] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection.
• Injectable depot forms may be made by forming microencapsule matrices of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of an antisense oligomer (e.g., eteplirsen), or pharmaceutically acceptable salt thereof, to polymer, and the nature of the particular polymer employed, the rate of release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations may also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
• an antisense oligomer e.g., eteplirsen
• biodegradable polymers such as polylactide-polyglycolide.
• the rate of release can be controlled. Examples of other biodegradable polymers include poly
• An antisense oligomer e.g., eteplirsen
• a pharmaceutically acceptable salt thereof can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, as described herein and known in the art.
• microemulsification technology may be utilized to improve bioavailability of lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S.
• microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation.
• Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8 glucose units, designated by the Greek letter a, b, or g, respectively.
• the glucose units are linked by a- 1,4-glucosidic bonds.
• all secondary hydroxyl groups at C-2, C-3) are located on one side of the ring, while all the primary hydroxyl groups at C-6 are situated on the other side.
• the external faces are hydrophilic, making the cyclodextrins water-soluble.
• the cavities of the cyclodextrins are hydrophobic, since they are lined by the hydrogen of atoms C-3 and C-5, and by ether-like oxygens.
• These matrices allow complexation with a variety of relatively hydrophobic compounds, including, for instance, steroid compounds such as 17a-estradiol (see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38: 1-3-113 (1994)).
• the complexation takes place by Van der Waals interactions and by hydrogen bond formation.
• the physico-chemical properties of the cyclodextrin derivatives depend strongly on the kind and the degree of substitution. For example, their solubility in water ranges from insoluble (e.g., triacetyl-beta-cyclodextrin) to 147% soluble (w/v) (G-2-beta- cyclodextrin). In addition, they are soluble in many organic solvents. The properties of the cyclodextrins enable the control over solubility of various formulation components by increasing or decreasing their solubility.
• Parmeter (I), et al. (U.S. Pat. No. 3,453,259) and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutral cyclodextrins.
• Other derivatives include cyclodextrins with cationic properties [Parmeter (II), U.S. Pat. No. 3,453,257], insoluble crosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), and cyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.
• the formulations contain micelles formed from an antisense
• oligomer e.g., eteplirsen
• at least one amphiphilic carrier in which the micelles have an average diameter of less than about 100 nm. More preferred embodiments provide micelles having an average diameter less than about 50 nm, and even more preferred embodiments provide micelles having an average diameter less than about 30 nm, or even less than about 20 nm.
• amphiphilic carriers While all suitable amphiphilic carriers are contemplated, the presently preferred carriers are generally those that have Generally-Recognized-as-Safe (GRAS) status, and that can both solubilize the compound of the disclosure and microemulsify it at a later stage when the solution comes into a contact with a complex water phase (such as one found in human gastro-intestinal tract).
• GRAS Generally-Recognized-as-Safe
• amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain straight chain aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene-glycolized fatty glycerides and polyethylene glycols.
• amphiphilic carriers include saturated and monounsaturated
• polyethyleneglycolyzed fatty acid glycerides such as those obtained from fully or partially hydrogenated various vegetable oils.
• oils may advantageously consist of tri-, di-, and mono-fatty acid glycerides and di- and mono-polyethyleneglycol esters of the corresponding fatty acids, with a particularly preferred fatty acid composition including capric acid 4-10, capric acid 3-9, lauric acid 40-50, myristic acid 14-24, palmitic acid 4- 14 and stearic acid 5-15%.
• Another useful class of amphiphilic carriers includes partially esterified sorbitan and/or sorbitol, with saturated or mono-unsaturated fatty acids (SPAN- series) or corresponding ethoxylated analogs (TWEEN-series).
• amphiphilic carriers may be particularly useful, including
• the delivery of an antisense oligomer may occur by use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the disclosure into suitable host cells.
• the compositions of the disclosure may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticle or the like.
• the formulation and use of such delivery vehicles can be carried out using known and conventional techniques.
• Liposomes consist of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar vesicles (SUVs) have a single membrane and typically range between 0.02 and 0.05 pm in diameter; large unilamellar vesicles (LUVS) are typically larger than 0.05 pm. Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 pm. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles.
• SUVs Small unilamellar vesicles
• Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 pm. Liposomes with several nonconcentric
• One aspect of the disclosure relates to formulations comprising liposomes
• an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof may be contained within, or adsorbed onto, the liposome bilayer of the liposome.
• An antisense oligomer e.g., eteplirsen
• a lipid surfactant e.g., glycerol, glycerol, or pharmaceutically acceptable salt thereof
• the liposome membrane is formulated to resist the disruptive effects of the active agent-surfactant aggregate.
• the lipid bilayer of a liposome contains lipids derivatized with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome, and extend from the exterior of the lipid bilayer into the surrounding environment.
• PEG polyethylene glycol
• a surfactant may be selected from any suitable aliphatic, cycloaliphatic or aromatic surfactant, including but not limited to biocompatible lysophosphatidylcholines (LPGs) of varying chain lengths (for example, from about C14 to about C20).
• LPGs biocompatible lysophosphatidylcholines
• Polymer-derivatized lipids such as PEG-lipids may also be utilized for micelle formation as they will act to inhibit micelle/membrane fusion, and as the addition of a polymer to surfactant molecules decreases the CMC of the surfactant and aids in micelle formation.
• Liposomes according to the disclosure may be prepared by any of a variety of techniques that are known in the art. See, e.g., U.S. Pat. No. 4,235,871; Published PCT applications WO 96/14057; New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104; Lasic DD, Liposomes from physics to applications,
• liposomes of the disclosure may be prepared by diffusing a lipid derivatized with a hydrophilic polymer into preformed liposomes, such as by exposing preformed liposomes to micelles composed of lipid-grafted polymers, at lipid concentrations corresponding to the final mole percent of derivatized lipid which is desired in the liposome.
• Liposomes containing a hydrophilic polymer can also be formed by homogenization, lipid-field hydration, or extrusion techniques, as are known in the art.
• an antisense oligomer e.g.
• micellar suspension of an antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt thereof is then used to rehydrate a dried lipid sample that contains a suitable mole percent of polymer- grafted lipid, or cholesterol.
• the lipid and an antisense oligomer suspension is then formed into liposomes using extrusion techniques as are known in the art, and the resulting liposomes separated from the unencapsulated solution by standard column separation.
• the liposomes are prepared to have substantially homogeneous sizes in a selected size range.
• One effective sizing method involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size; the pore size of the membrane will correspond roughly with the largest sizes of liposomes produced by extrusion through that membrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).
• reagents such as DharmaFECT® and Lipofectamine® may be utilized to introduce polynucleotides or proteins into cells.
• release characteristics of a formulation of the disclosure depend on the encapsulating material, the concentration of encapsulated drug, and the presence of release modifiers.
• release can be manipulated to be pH dependent, for example, using a pH sensitive coating that releases only at a low pH, as in the stomach, or a higher pH, as in the intestine.
• An enteric coating can be used to prevent release from occurring until after passage through the stomach.
• Multiple coatings or mixtures of cyanamide encapsulated in different materials can be used to obtain an initial release in the stomach, followed by later release in the intestine.
• Release can also be manipulated by inclusion of salts or pore forming agents, which can increase water uptake or release of drug by diffusion from the capsule.
• Excipients which modify the solubility of the drug can also be used to control the release rate.
• Agents which enhance degradation of the matrix or release from the matrix can also be incorporated. They can be added to the drug, added as a separate phase (i.e., as particulates), or can be co-dissolved in the polymer phase depending on the compound. In most cases the amount should be between 0.1 and thirty percent (w/w polymer).
• Types of degradation enhancers include inorganic salts such as ammonium sulfate and ammonium chloride, organic acids such as citric acid, benzoic acid, and ascorbic acid, inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, zinc carbonate, and zinc hydroxide, and organic bases such as protamine sulfate, spermine, choline, ethanolamine, diethanolamine, and triethanolamine and surfactants such as Tween® and Pluronic®.
• Pore forming agents which add microstructure to the matrices i.e., water soluble compounds such as inorganic salts and sugars
• the range is typically between one and thirty percent (w/w polymer).
• Hydrophilic polymers suitable for use in the liposomes are those which are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e., are biocompatible).
• Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), poly glycolic acid (also termed polyglycolide), a polylactic-poly glycolic acid copolymer, and polyvinyl alcohol.
• polymers have a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, or from about 300 daltons to about 5,000 daltons.
• the polymer is poly ethyleneglycol having a molecular weight of from about 100 to about 5,000 daltons, or having a molecular weight of from about 300 to about 5,000 daltons.
• the polymer is polyethyleneglycol of 750 daltons (PEG(750)).
• Polymers may also be defined by the number of monomers therein; a preferred embodiment of the disclosure utilizes polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).
• polyhydroxypropyl methacrylamide polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
• a formulation of the disclosure comprises a
• biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co- caprolactone), polysaccharides, proteins, polyhyaluronic acids, poly cyanoacrylates, and blends, mixtures, or copolymers thereof.
• Uptake can also be manipulated by altering residence time of the particles in the gut. This can be achieved, for example, by coating the particle with, or selecting as the encapsulating material, a mucosal adhesive polymer.
• a mucosal adhesive polymer examples include most polymers with free carboxyl groups, such as chitosan, celluloses, and especially polyacrylates (as used herein, polyacrylates refers to polymers including acrylate groups and modified acrylate groups such as cyanoacrylates and methacrylates).
• An antisense oligomer e.g., eteplirsen
• pharmaceutically acceptable salt e.g., eteplirsen
• an implant may be coated or otherwise treated with an oligomer.
• an oligomer for example, hydrogels, or other polymers, such as
• biocompatible and/or biodegradable polymers may be used to coat an implant with the compositions of the disclosure (i.e., the composition may be adapted for use with a medical device by using a hydrogel or other polymer).
• Polymers and copolymers for coating medical devices with an agent are well-known in the art.
• implants include, but are not limited to, stents, drug-eluting stents, sutures, prosthesis, vascular catheters, dialysis catheters, vascular grafts, prosthetic heart valves, cardiac pacemakers, implantable cardioverter defibrillators, IV needles, devices for bone setting and formation, such as pins, screws, plates, and other devices, and artificial tissue matrices for wound healing.
• Example 1 High-dose eteplirsen treatment in the h DMD D52 mdx mouse model.
• Eteplirsen was tested in a humanized DMD mdx mouse model, wherein the
• human DMD with exon 52 deletion-YAC transgene (h DMD L52 mouse) was integrated into mouse chromosome 5.
• the model is described in Hoen et al,“Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene,” J. Biol. Chem., 2008, 283(9):5899-5907, disclosure of which is incorporated herein in its entirety.
• exon 52 in the human DMD gene produces an out of frame pre- mRNA.
• the h DMD D52 mouse is amenable to exon 51 skipping drugs.
• eteplirsen is designed to bind to exon 51 of dystrophin pre-mRNA in the h DMD L52 mouse, resulting in exclusion of this exon during mRNA processing. Skipping of exon 51 by eteplirsen is anticipated to restore the open reading frame of dystrophin mRNA during protein translation. This is intended to allow for the production of an internally shortened dystrophin protein that localizes to the muscle membrane and can protect muscle from damage, as well as improve force in dystrophic muscle fibres.
• mice were treated with once weekly (QW) intravenous injections of vehicle or eteplirsen for 1 (500 or 960 mg/kg), 4 (500 or 750 mg/kg), and 8 weeks (500 or 750 mg/kg) or 8 weeks.
• QW once weekly
• the plasma exposures at 500, 750, and 960 mg/kg doses in mice are equivalent to 100, 150, and 192 mg/kg plasma exposures in humans. This is based on 5: 1 dose/exposure relationship difference between mouse and human.
• Dystrophin production in hDMD D52 mdx mice was measured according to the following method. Protein lysates prepared in NCH buffer were diluted to 0.2pg/pl in 0. lx sample buffer provided with the Jess kit. A wild type standard curve was prepared using pooled wild type lysates diluted in pooled dystrophic lysate, at a concentration of 0.2pg/pl in O. lx sample buffer. 4pl of each sample and lpl of prepared fluorescent 5x master mix were added to a PCR plate. The plate was then incubated at 95 °C for 5 minutes, and then placed on ice. 3m1 sample was then loaded onto the Jess plate, in the row indicated by the manufacturer. The rest of the plate was prepared according to the manufacturer’s instructions, using milk free antibody diluent.
• the primary antibody cocktail was prepared by diluting dystrophin antibody
• the secondary antibody cocktail was prepared by diluting 20x NIR anti-mouse antibody (obtained from Protein Simple; Beekman et al,“Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy,” PLOS One, Apli 11, 2018, content of which is incorporated herein in its entirety) in anti-rabbit chemiluminescent antibody (Protein simple). Once the plate was loaded, it was placed in the Jess, and a 66-440kDa 25 capillary module was inserted into the instrument. The assay was then run using the programs for both chemiluminescence and
• dystrophin was normalized to actinin by dividing the peak area for dystrophin (308kDA) by the peak area for actinin (106kDa) for each
• Percent WT was calculated by fitting a line to the standard curve and using the equation of that line to find the percent of dystrophin compared to the humanized DMD WT mouse.
• Figure 2 demonstrates that eteplirsen increases dystrophin production in h DMD D52 mdx mice.
• the Grip Strength test measures the maximal peak force developed by a rodent.
• the peak force is measured in grams and is obtained by the operator drawing the mouse along a straight line over a grid leading away from the sensor it is attached to. The animal will release at the end of the grid and a maximum force measurement is obtained. Three measurements are taken per each day of testing and averaged to obtain results. Results are normalized to the animals body weight. Mice are tested at onset of study to determine a baseline and train mice to the apparatus. Figure 3 shows that eteplirsen improves function in hDMD D52 mdx mice.
• IV intravenously
• SC subcutaneously
• Group 1 animals were administered Vehicle Control.
• Groups 2-4 were administered Vehicle Control.
• eteplirsen administered eteplirsen at dose levels of 5-320 mg/kg as IV bolus.
• Group 5 was administered eteplirsen as SC dose at 320 mg/kg dose.
• a 320 mg/kg dose in humans is predicted to provide AUC and C max PK that are equivalent to the AUC and C max PK provided by 320 mg/kg in NHP. Based on the dose-linear exposure relationship observed in NHP, the human 200 mg/kg dose is expected to exhibit similar AUC and Cmax PK. .
• Eligible patients are boys between 7 and 13 years of age (inclusive), with out-of- frame deletions of the DMD gene that could be corrected by skipping exon 51.
• Patients have achieved a mean 6-minute walk test (6MWT) distance of >300 and ⁇ 450 meters (without assistance) at both the screening and baseline visits.
• 6MWT mean 6-minute walk test
• Patients have intact right and left biceps muscles (the preferred biopsy site) or an alternative upper arm muscle group that will allow for sufficiently sized (1 cm 3 ) muscle biopsies to be obtained prior to and on treatment.
• Patient also have been on a stable dose or dose equivalent of oral corticosteroids for at least 24 weeks prior to randomization, and the dose is expected to remain constant (except for modifications to accommodate changes in weight) throughout the study.
• Patients also have stable pulmonary function (forced vital capacity >50% of predicted and no requirement for nocturnal ventilation) and pulmonary function is unlikely to decompensate significantly over the duration of the study.
• SEQ ID NO: l is supplied by Sarepta Therapeutics, Inc. in single-use vials of phosphate- buffered saline (100 mg/ml). Eteplirsen is reconstituted with 150 ml normal saline and infused over 60 minutes. Placebo is administered during the first 24 weeks of the study. Placebo is supplied as identical vials of phosphate-buffered saline and administered in the same manner as eteplirsen.
• the objective of this study is to evaluate the safety and tolerability of weekly IV doses of 100 and 200 mg/kg of eteplirsen, and to evaluate the pharmacokinetics of 100 and 200 mg/kg of eteplirsen.
• a cohort of 4 eligible DMD patients is treated with eteplirsen IV weekly at a dose of 100 mg/kg followed by 200 mg/kg, for 4 weeks at each dose level; each 4-week treatment period may be extended based on patient enrollment. After each 2-week treatment period has been completed by 4 patients, safety and tolerability data is reviewed to determine if the patients will continue dosing for 2 more weeks.
• the cohort may be expanded by 2 additional patients at each dose level, with initiation of dosing after 2 weeks of treatment within the applicable 4-week treatment period.
• the available data is evaluated to determine if 100 mg/kg or both 100 and 200 mg/kg weekly IV doses is (are) adequately safe and tolerated in DMD patients such that the Double-blind portion of the study can begin. Patients in the Open-label Dose Escalation continue treatment with the selected high dose as a distinct cohort.
• Finding is not conducted, and patients are randomized in a 1 :2 ratio to 30 or 100 mg/kg doses for Dose Comparison.
• Example 4 DMD patient myotube assay with high-dose golodirsen.
• the cell model used is a muscle cell line isolated from a DMD patient with an exon 52 deletion (DMD Del52) and immortalized by the Institute of Myology using a method shown to preserve the essential primary skeletal muscle characteristics of the cells (Mamchaoui et al, "Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders," Skeletal Muscle, 2011, 1(1):34; Thorley et al,“Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines," Skeletal Muscle, 2016, 6(1):43; content of both references is incorporated herein in its entirety).
• Myoblasts were isolated from the paravertebral muscles of a 16 year old healthy donor and 16 year old DMD patient with a deletion in exon 52 and immortalized by the Institute of Myology by ectopic expression of hTERT and CDK4 as previously described (Mamchaoui et al, "Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders," Skeletal Muscle, 2011, 1(1):34).
• Cells were maintained in proliferation medium containing 1 volume medium 199, 4 volumes Dulbecco’s modified Eagle’s medium (DMEM), 20% fetal bovine serum, 50 pg/ml gentamycin, 25 pg/ml fetuin, 0.5 pg/ml bFGF, 5 ng/ml EGF, 0.2 pg/ml dexamethasone, 5 pg/ml insulin on tissue culture plates coated with 1% collagen I and 0.5% MaxGel (Sigma-Aldrich E0282) at 50 ul/cm2 for 3 hr at 37°C.
• DMEM Dulbecco’s modified Eagle’s medium
• Positive and negative control compounds were included on each plate for assay quality control.
• the positive control was 30 pM SRP-5051, lot # RD00128-17 and the negative control 30 pM RC-1001 (MZ-194-170). All compounds were dissolved in sterile water and the concentration was confirmed by spectrophotometry prior to assay.
• Myoblasts were plated in proliferation medium at 6000 cells/well in a 96-well clear bottom imaging plate (Perkin Elmer #6055300) coated with 1% collagen I and 0.5% MaxGel (Sigma-Aldrich E0282) at 50 pl/well for 3 hr at 37 °C.
• Cells were stained with primary antibodies rabbit anti-MyoD (1 : 100, Fisher Scientific, # NC0819717), mouse anti-Dystrophin MANDRA1 (IgGl(7A10), Santa Cruz, #sc-47760), mouse anti-Dystrophin (IgG2ak, MANDYS106, EMD Millipore, # MABT827), mouse anti-Myosin heavy chain (IgG2B, 1 : 1000, R&D System, #MAB4470) and secondary antibodies Alexa Fluor 488 donkey anti-rabbit (1 : 1000, Life Technologies, #A21206), Alexa Fluor 555 goat anti-mouse IgG2a (l: 1000, Life Technologies, #A21137), Alexa Fluor 555 goat anti-mouse IgGl (1: 1000, Life Technologies, # A21127) and Alexa Fluor 647 goat anti-mouse IgG2b (1: 1000, Life Technologies, #A21242). Nuclei were stained with DAPI (1 pg/
• Golodirsen was administered to male cynomolgus monkeys by IV bolus injection once weekly for 12 weeks, at dose levels of 0 (vehicle), 5, 40, or 320 mg/kg, followed by a 4 week recovery period.
• a 320 mg/kg dose in humans is predicted to provide AUC and C max PK that are equivalent to the AUC and C max PK provided by 320 mg/kg in NHP.
• the human 200 mg/kg dose is expected to exhibit similar AUC and C max PK.

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